Neuronal development requires the unfoldment of a highly complex differentiation program. Deciphering the regulatory code underlying this program has direct relevance for CNS disease, since many disorders arise from disruption of one or more neuronal differentiation events. In particular, it is now clear that the interplay between transcription factors and dynamic changes in chromatin structure during neuronal development is altered in several nervous system disorders. As neurons mature, they pass through successive maturation stages in which unique subsets of genes must be turned on and off in the proper sequence and timing. Altering this temporal program can result in serious developmental defects. Individual transcription factors often function during multiple stages of neuronal differentiation, and what determines the temporal patterning for the distinct subsets of genes they control at different steps is not understood. The central goal of this proposal is to define the critical timekeeping mechanisms that ensure the sequential ordering of gene transcription during neuronal differentiation. To address this, we are exploring the temporal regulation of a neuron-specific gene during development. In this proposal, we will examine key interactions between trans-activators, trans-repressors and chromatin structure that collaborate in determining the regulation of the transcriptional timer involved. We will examine temporal interactions of different trans-factors and chromatin modifiers with native chromatin as well as various chromatin modifications occurring within the target gene locus, both in primary neurons and within the CNS. Promoter analyses also will be performed in primary neuronal cultures and in vivo using transgenic approaches. In addition, specific gene knockouts for candidate timekeepers will be examined to determine whether trans-factor disruption alters the transcriptional timer in vivo. Results from these studies will provide insight into how different phases of neuronal development are coordinated to ensure appropriate maturation and function. They also will identify important developmental regulators that may underlie CNS disorders. PUBLIC HEALTH RELEVANCE Defects in genes that control the proper formation of the nervous system frequently giver is to severe neurological disorders. Our studies will identify key mechanisms that determine the appropriate timing of nervous system development as well as candidate CNS disease-related genes involved in this process.